Methods in molecular biology
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The increase in the number of Web-based resources on posttranslational modification sites (PTMSs) in proteins is accelerating. This chapter presents a set of computational protocols describing how to work with the Internet resources when dealing with PTMSs. The protocols are intended for querying in PTMS-related databases, search of the PTMSs in the protein sequences and structures, and calculating the pI and molecular mass of the PTM isoforms. Thus, the modern bioinformatics prediction tools make it feasible to express protein modification in broader quantitative terms.
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Gene editing has great therapeutic impact, being of interest for many scientists worldwide. Clustered regularly interspaced short palindromic repeats (CRISPR) technology has been adapted for gene editing to serve as an efficient, rapid, and cost-effective tool. ⋯ The gRNA targets the genome site to be edited, giving great importance to its design to obtain increased efficiency and decreased off-target events. In this chapter, we describe different tools to design suitable gRNAs for a variety of experimental purposes.
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DNA methylation is a conserved epigenetic modification of animal genomes, but genome methylation patterns appear surprisingly diverse in insects. Whole-genome bisulfite sequencing (WGBS) represents a sensitive and robust method for the characterization of genome-wide methylation patterns at single-base resolution. Here, we describe a step-by-step protocol for the generation and analysis of WGBS datasets using standard Illumina sequencing platforms. In comparison to whole-genome sequencing, WGBS has additional caveats that require particular attention and are highlighted in this chapter.
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DNA methylation changes are dynamic processes which occur at cytosines of CpG dinucleotides and contribute to normal development but also to diseases. DNA methylation changes are most effective in promoters and enhancers, the former frequently being CpG-rich and the latter, in contrast, CpG-poor. Many genome-wide methods for DNA methylation analysis interrogate predominantly CpG-rich regions and, hence, spare enhancers and other potentially important genomic regions. ⋯ In a subsequent step, the non-covalently bound adapter oligonucleotide needs to be replaced by a novel oligonucleotide to provide the proper adapter sequence for the reverse strand in paired-end sequencing. The presented protocol describes an improved, simplified version of TWGBS where the inefficient oligo-replacement is circumvented by usage of a sequencing-compatible transposase-adapter complex. Consequently, genomic DNA of only a few hundred human cells is required to interrogate the complete human DNA methylome.
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Rapid evaluation of the CRISPR gRNA activity is an essential step of employing the technology in editing genes. Through machine learning strategy, the rule sets for in silico designing gRNAs with high activity has greatly improved. However, there are still discrepancies between different prediction rule sets, and between the predicted and actual gRNA activities. ⋯ We had previously developed a dual-fluorescent surrogate system, called C-Check, which based on single-strand annealing repair of the DNA double-strand breaks introduced by CRISPR-Cas9 to generate a functional EGFP. The system offers a tool for rapid functional evaluation of CRISPR gRNA activity, as well as for enrichment of gene edited cells. In this chapter, we will give a step-by-step instruction on the design, generation, and application of the C-Check system for quantifying gRNA activities.